Generally, living cells are not permeable to macromolecules such as proteins or nucleic acids. The fact that only small-size molecules can permeate through the membrane of living cells at very low rates has restricted the researches to develop drugs to cure, prevent or diagnose diseases using macromolecules including, for example, proteins and nucleic acids. On the other hand, because most of the substances manufactured to cure, prevent or diagnose certain diseases have to be delivered into the cytosol with effective amounts, there have been several methods developed delivering those substances from the cell surface of the target cell into the cell.
Methods used to deliver macromolecules into cells include electroporation, cytosol fusion using liposomes, highly concentrated projection technique of a projectile coated with DNA on surface, calcium-phosphorous-DNA precipitation method, DEAE-dextran transfection, infection with modified viral nucleic acids, and direct microinjection into a single cell, etc. Moreover, there have been attempts to deliver macromolecules using nanoparticles in vitro and in vivo these days, but it is only in its beginning step technologically and clinically. Furthermore, these methods can deliver macromolecules to only a few of the target cells, and its efficiency is not sufficient enough to be applied clinically. Also, most of these methods cause side effects on the other cells.
In this regard, the demand for the development of a novel method of delivering biologically active macromolecules into the target cells effectively in vivo and in vitro is increasing (L. A. Sternson, Ann. N.Y. Acad. Sci., 57, 19-21(1987)). Chemical addition of lipid peptide (P. Hoffmann et al. Immunobiol., 177, 158-170(1988)) or use of base polymers such as polylysine or polyarginine (W-C. Chen et al., Proc. Natl. Acad. Sci., USA. 75, 1872-1876(1978)) were provided. In addition, it was reported that folate of a transporter was transferred into a cell in the form of folate-conjugate. But it has not been confirmed yet that the folate was transduced into the cytosol. Also, Pseudomonas Exotoxin is known as a transporter (T. I. Prior et al., Cell. 64, 1017-1023(1991)). However, the effects of biologically active macromolecules delivered into the cytosol and their general application have not been clearly verified yet. Therefore, an effective method of transducing biologically active macromolecules into cytosol and nucleus of living cells is highly demanded.
In addition, the efficient delivery of DNA/RNA as well as macromolecules, such as proteins, into cells in vivo and in vitro is considered as to be one of the essential technique required in the field of biotechnology and applied medical science. The delivery of DNA/RNA into cells acts as a decisive factor for gene therapies, for studying the revealation of the function of a protein encoded by the gene in vivo and in vitro, and for the development of novel remedies using DNA/RNA. However, since DNA/RNA cannot permeate the cell membrane efficiently, it is very important for using genes in basic and clinical researches to improve the permeability.
For this reason, liposomes, nanomolecules, and viral vectors etc. are developed to deliver DNA and/or RNA into a cell in vitro and in vivo, and the possibilities of the use thereof were examined and investigated. However, concerning beneficial-effects and side effects, it has numerous problems to be resolved. In particular, regarding liposomes, since the side effects against cells and the cytotoxicity are very serious, their application was limited to basic researches. As for nanomolecules, though it has been receiving attentions these days, the decomposition of carrier particles in vivo, the poor efficiency of transduction and the immunological responses elicited by the molecules should be studied further and should be resolved. As for retroviruses, it has a problem in that it cannot infect undeviding cells. Adenovirus or adeno-associated virus vector also has a very limited clinical application. Furthermore, these two types of viral vectors may elicit immune responses against the other viral proteins, so its treating efficiency has many doubts. Therefore, a new way to transduce DNA/RNA into cells efficiently and less detrimentally is needed.
Meanwhile, proteins regulating physiological phenomena in vivo, are produced by bacteria, such as E. coli, as a form of recombinant protein, and have been employed in the treatment of numerous diseases. The proteins, which were synthesized in bacteria, however, were known as to be inefficient in folding structures and functions in comparison to the naturally folded proteins in vivo. Thus, there have been lots of attempts to produce proteins in yeasts, insect cells or animal cells, and to make the proteins produced in bacteria refolded using transgenic animals. However, these methods require further studies and full understanding on many molecular cell biological intermediate steps, and their transduction efficiencies are very low. And they are not cost effective.
Several PTDs (Protein Transduction Domain) have been reported as a result of this demand. Among them, Tat protein, which is a Human Immunodeficiency Virus-1 (HIV-1) viral protein, has been mostly well studied. The Tat protein was known to operate more efficiently when containing amino acids 47 to 57 (YGRKKRRQRRR), where positive charged amino acids are concentrated, than containing full-length 86 amino acid protein (Fawell S. et al. Proc. Natl. Acad. Sci. USA 91, 664-668(1994)). Other examples of PTDs are amino acids 267 to 300 of Herpes Simplex Virus type 1 protein (HSV-1) (Elliott G. et al. Cell, 88, 223-233(1997)), amino acids 339 to 355 of Antennapedia (ANTP) protein of Drosophila (Schwarze S. R. et al. Trends Pharmacol Sci. 21, 45-48(2000)), and artificial combination of positively charged amino acids. Regarding the PTDs mentioned above, we, inventors, found that they contained lysine and arginine abundantly, wherein the arginine was considered to play a great role in the transduction of biomolecule into cells. And it was supported by the published document that disclosed transduction activities of artificial peptides consisting of positively charged amino acids. (Laus R. et al. Nature Biotechnol. 18, 1269-1272(2000)).
With regard to the transduction mechanism of macromolecules into cells when using PTDs, there are 2 (two) hypothesises. The first is that PTDs ruin the plasma membrane, and trasmit the molecules across it. The second one is that PTD uses the plasma membrane to form a new vesicle that can carry the molecules into the cells. Moreover, there are suppositions that PTD has structural features that can form new channels in the membrane (Becker-Hapak M. et al, 2001, Jul:24(3):247-256).
However, experiments with artificially combined amino acids with 12 arginines and 12 lysines suggested that the hypothesis that the existance of lysine and arginine at specific positions induced the formation of new channels might be wrong (Rothbard J B, et al, Nature Med. 200 Nov:6(11): 1253-1257). In addition, considering the facts that only the proteins, which were bound covalently or non-covalently to PTDs, were transferred through the cell membrane, the hypothesis, that PTDs ruin the plasma membrane and translocate the molecules across it, was not acceptable. Furthermore, according to our studies, PTDs represented transduction abilities both at 37° C. and at 4° C., which suggested that PTD neither made new channels nor made new vesicles.
Recently, as a new type of PTD, MTS was developed. Its amino acid sequence was synthesized based on the signal peptide of FGF (Fibroblast Growth Factor), while amino acids of the signal peptide were known to have features quite different from those of PTD amino acids as follows. (a) 3-5 numbers of arginines or lysines exist non-continuously together with serine or threonine, and there are no glutamic acid and aspartic acid, (b) one or more basic amino acids, and 6-12 numbers of hydrophobic amino acids, (c) serine, threonine, or small size hydrophobic amino acid exists abundantly, and glutamine, aspartic acid are present in small amount, (d) 10 random amino acids are present between 1 or 2 basic amino acid(s) that are gathered together. Thus, the MTS is thought to have different features in comparison to the ordinary PTDs. That is, the MTS has different amino acid combination.
In this regard, we have tried to find out a new machinery as to the PTD transduction and decisive factors to the amino acid combination consisting of PTDs, based on the discovered features of the two types of PTDs above mentioned and based on the results of our preliminary researches. The followings are two new hypothesis derived from the decisive factors and requisites for the development of a new type of PTD of the present invention: 1) considering that i) unfolded proteins are transduced more efficiently than completely folded ones, and that ii) once the unfolded proteins are transmitted into organelles or cells they are not eliminated from the organelles or cells, and that iii) PTDs do not utilize receptors to perform endocytosis or phagocytosis, it seems that the PTDs use channels present on the cell surface. Accordingly, hydrophobic amino acids, such as alanine and valine, are highly demanded; 2) Since PTDs transmit molecules into the nucleus efficiently, its function may be similar to transcription factors. Therefore, PTDs would be found frequently in transcription factors. And the PTDs may use channels similar to translocons that transmit proteins into organelles.
Based on these two hypothesis and requisites, we searched the gene bank. With the factor that the conventional PTDs have lysines and arginines abundantly, we selected about 10,000 primary candidate genes. Among them, 500 genes were chosen by applying the requisites of signal peptide, and 100 genes of the 500 genes were confirmed as to have alanine and valine. Finally, 20 genes were selected by applying the factors required for the transcription factors, and then the transduction efficiencies were tested therefor. In addition, fusion proteins of each of these candidate PTDs and β-galactosidase were expressed and purified, and the functions of these proteins were detected using Jurkat T cells. As a consequence, amino acid sequence 558 to 566 of Sim-2 (number of gene bank: U80456), a human transcription factor, was found to have unexpectedly significant transduction ability and was named Sim-2 BTM (Biomolecule Transduction Motif).
Thus, we completed the present invention with the findings that amino acids 558 to 566 of Sim-2 have significantly excellent features as an intracellular biomolecule transduction peptide, thereby any target proteins, nucleic acids, lipids, carbohydrates and chemical compounds can be efficiently delivered into cytosol and nucleus in vivo and in vitro. Furthermore, we verified that the desired macromolecules could be transduced into specific organs and/or cells in vivo and in vitro, by employing ecto domain of the ligand, which binds to the receptor present on the surface of the cells or the organs to which the macromolecules are transduced, MMP cleavage site and Sim-2 BTM. Furthermore, we found that expression vector having 5 (five) successive DNA/RNA sequences, which specifically bind to DNA/RNA binding domains, could be transduced in vivo and in vitro into the specific organs or cells, using the Sim-2 BTM and using the DNA/RNA binding domains (DBD and/or RBD) that bind to the DNA/RNA to be transduced, wherein the expression vector could include regulatory elements comprising a promoter that induces gene expression selectively in specific organs, tissues or cells. Furthermore, according to the present invention, it is possible to reform the structure of recombinant protein to have the same folding structure and functions as those found naturally, by transferring the proteins synthesized in bacteria to the suitable animal cells and isolating them.